Our goal was to examine changes in muscle acetyl-CoA, long-chain acylcarnititie (LCA), and short-chain acylcamitine (SCA) concentrations in response to hyrerinsulinernic-hyperglycemia Seven healthy volunteers had a muscle biopsy taken from the lateral aspect of the vastus lateralis muscle approximately 15 cm above the patella, in the morning after they had fasted overnight (basal). Immediately following the basal period a hyperinsulinemic-hyperglycemic clamp (insulin=275 uU/mL, glucose= 140 mg/dL) was started and continued for 5 h. Plasma FFA concentration was kept constant by means of variable infusions of Intralipid and heparin. A second muscle biopsy was taken from the same incision site at the end of the study. Carbohydrate oxidation significantly increased during the clamp (5.1+1.3 to 26.8+3.0 umol/kg.min, P<0.0l). However, despite the significant increase in pyruvate flux through pyruvate dehydrogenase, we did not observe any significant change in the acetyl-CoA-to-CoA ratio, suggesting that the contribution of B-oxidation-derived acetyl-CoA decreased during the clamp. The significant decrease in SCA concentration (5O64+l776 to 2192+448 nmol/g dry wt., P<0.05), suggested that fatty acid oxidation was limited at a step preceding B-oxidation. This was confirmed by the observed decrease in LCA concentration during the clamp (855+270 to 377+83 nmol/g dry wt, P<O.05), in concert with the notion that high pyruvate flux blocks fatty acid entry into the mitochondria via inhibition of carnitine palmitoyltransferase I. (Supported by NIH grants DK-34817 and DK-46017 and Shriners Hospital grant 15849.)